Mitochondrial NADH fluorescence is enhanced by complex I binding.
Mitochondrial NADH fluorescence is enhanced by complex I binding.
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DOI：
10.1021/bi800307y
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发表时间：
2008-09-09
期刊：
影响因子：
2.9
通讯作者：
Balaban, Robert S.
中科院分区：
文献类型：
--
作者：
Blinova, Ksenia;Levine, Rodney L.;Boja, Emily S.;Griffiths, Gary L.;Shi, Zhen-Dan;Ruddy, Brian;Balaban, Robert S.
线粒体NADH荧光已成为一种有用的工具，在体外和体内评估线粒体能量。通过延长荧光寿命（EFL），线粒体NADH荧光在基质中增强数倍。然而，负责NADH EFL的实际结合位点是未知的。我们测试的假设，NADH结合到复合物I是线粒体NADH荧光增强的重要来源。为了检验这一假设，在纯化的蛋白质和整个猪心线粒体蛋白质组的天然凝胶中评价复合物I结合对NADH荧光效率的影响。为了避免这些制剂中的NADH氧化，我们在专门设计的装置中在缺氧条件下进行了结合实验。纯化的完整复合物I在天然凝胶中增强NADH荧光约10倍。然而，在变性的单个复合物I亚基蛋白中没有检测到增强。在整个线粒体蛋白质组的Clear和Ghost天然凝胶中，NADH荧光增强定位于在氧存在下发生NADH氧化的区域。抑制剂和质谱研究表明，荧光增强是特定的复合物I蛋白质。在该测定系统中，在基质蛋白的生理摩尔分数下，未检测到MDH或其他脱氢酶的荧光增强。这些数据表明，与复合物I相关的NADH显著地贡献于整体线粒体NADH荧光信号，并为线粒体NADH荧光和代谢状态的良好建立的密切相关性提供了解释。
Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10 fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state.
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DOI：
10.1196/annals.1380.013
发表时间：
2006-01-01
期刊：
INTERACTIVE AND INTEGRATIVE CARDIOLOGY
影响因子：
--
作者：
Balaban, Robert S.
通讯作者：
Balaban, Robert S.
影响因子：
4.1
作者：
CHANCE, B;PACKER, L
通讯作者：
PACKER, L
影响因子：
2.9
作者：
DUCKWORTH, HW;TONG, EK
通讯作者：
TONG, EK
影响因子：
2.9
作者：
ESTABROOK, RW
通讯作者：
ESTABROOK, RW
影响因子：
3.4
作者：
Joubert, F;Fales, HM;Balaban, RS
通讯作者：
Balaban, RS